Various flow cytometers and microfluidic systems exist for the purpose of analyzing and sorting particles. Each of these instruments has various shortcomings relating to their ability to maintain accurate sort actions. Jet-in-air flow cytometers are commonly used for the purpose of sorting particles based on detected characteristics. The operation of a jet-in-air flow cytometer may involve producing a coaxial fluid stream in a nozzle. The coaxial fluid stream has a core stream of sample, which includes the particles or interest, and an outer stream of sheath fluid. The sheath fluid provides a means for positioning particles and preventing clogging in the nozzle, as well as, for providing conductive medium suitable for retaining an applied charge.
The coaxial fluid stream may be perturbed with an oscillator, such as a piezoelectric crystal, resulting in the formation of droplets downstream of a nozzle. The droplets may contain individual particles or a small group of particles. Based on a desired sort action, each droplet may be charged just prior to separating from the fluid stream at a break off point. The appropriate time for applying this charge is known as the drop delay. As droplets may be formed at a rate of between about 20,000 per second and 200,000 per second, the drop delay must be very precisely calculated.
Historically, the drop delay was determined through an iterative series of protocols largely consisting of trial and error test streams having varying drop delays. Beads or particles were run through test streams and collected in puddles. The number of beads or particles collected in each puddle provided an indication of the drop delay. Such manual protocols are time intensive and may be lacking in the precision required to achieve extremely accurate sort decisions and are incapable of real time verifications or adjustments.
U.S. Pat. No. 6,248,590 describes an attempt to monitor the drop delay with the use of a single camera for imaging a portion of the fluid stream, or with multiple cameras for imaging separate portions of the fluid stream, such as for determining the speed of particles at the nozzle and at the speed of particles at a drop off point. From this information an approximation is derived utilizing an exponential decay model. However, the formation of droplets may not be so easily predictable and because only a portion of the stream is monitored, upstream changes may not be detected reliably.
U.S. Patent application publication 2001/02218892 provides a camera mounted on a movable stage for taking multiple images of a fluid stream. The images are then stitched together and the widths are determined. From this information a number of droplets, peaks, and periods between the inspection zone and the drop delay maybe determined. This configuration provides for means of producing a composite image of the entire fluid stream, but is lacking the ability to monitor the fluid stream in real time because the camera must traverse the fluid stream at a rate thousands of times slower than the fluid stream itself. Further, a stitched together image may not accurately reflect the stream at any particular time and is time consuming to produce. Changes in operating conditions, such as oscillator frequency, oscillator amplitude, temperature, surface tension, and harmonic conditions may vary the stream during the time in which the individual images are being captured. Positional changes in the excitation source or the droplet break off point during operation, upon start up, or during other changes cannot quickly be realized by the described system because a series of images must first be captured and then stitched together.